We have observed rapid and extensive depletion of cellular energy stores by ⁹-tetrahydrocannabinol (THC) in the pulmonary transformed cell line, A549. ATP levels declined dose-dependently with an IC₅₀ of 7.5µg/ml THC following 24 hr exposure. Cell death was observed only at concentrations above 10 µg/ml. Studies using JC-1, a fluorescent probe for mitochondrial membrane potential, revealed diminished mitochondrial function at THC concentrations as low as 0.5 µg/ml. At concentrations of 2.5 or 10 µg/ml THC, a decrease in mitochondrial membrane potential were observed as early as 1 hr after THC exposure. Mitochondrial function remained diminished for at least 30 hr following THC exposure. Flow cytometry studies on cells exposed to particulate smoke extracts indicated that JC-1 red fluorescence was 5-fold lower in cells exposed to marijuana smoke extract relative to cells exposed to tobacco smoke extract. Comparison with a variety of mitochondrial inhibitors demonstrated that THC produced effects similar to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), suggesting uncoupling of electron transport. Loss of red JC-1 fluorescence by THC was suppressed by cyclosporin A, suggesting mediation by the mitochondrial permeability transition pore. This disruption of mitochondrial function was sustained for at least 24 hr following removal of THC by extensive washing. These results suggest that exposure of the bronchopulmonary epithelium to THC may have important health and physiological consequences.